Role of Residual Monomers in the Manifestation of (Cyto)toxicity by Polystyrene Microplastic Model Particles.

Polystyrene (PS) is an important model polymer for the investigation of effects of microplastic (MP) and nanoplastic (NP) particles on living systems. Aqueous dispersions of PS MP or NP contain residual monomers of styrene. In consequence, it is not clear if the effects observed in standard (cyto)toxicity studies are evoked by the polymer (MP/NP) particle or by residual monomers. We addressed that question by comparing standard PS model particle dispersions with in-house synthesized PS particle dispersions. We proposed a rapid purification method of PS particle dispersions by dialysis against mixed solvents and developed a simple method of UV-vis spectrometry to detect residual styrene in the dispersions. We found that standard PS model particle dispersions, which contain residual monomers, exerted a low but significant cytotoxicity on mammalian cells, while the in-house synthesized PS, after rigorous purification to reduce the styrene content, did not. However, the PS particles per se but not the residual styrene in both PS particle dispersions resulted in immobilization of Daphnia. Only by using freshly monomer-depleted particles, will it be possible in the future to assess the (cyto)toxicities of PS particles, avoiding an otherwise not controllable bias effect of the monomer.

The complexity of micro- and nanoplastic research in the genus Daphnia - A systematic review of study variability and a meta-analysis of immobilization rates.

In recent years, the number of publications on nano- and microplastic particles (NMPs) effects on freshwater organisms has increased rapidly. Freshwater crustaceans of the genus Daphnia are widely used in ecotoxicological research as model organisms for assessing the impact of NMPs. However, the diversity of experimental designs in these studies makes conclusions about the general impact of NMPs on Daphnia challenging. To approach this, we systematically reviewed the literature on NMP effects on Daphnia and summarized the diversity of test organisms, experimental conditions, NMP properties and measured endpoints to identify gaps in our knowledge of NMP effects on Daphnia. We use a meta-analysis on mortality and immobilization rates extracted from the compiled literature to illustrate how NMP properties, study parameters and the biology of Daphnia can impact outcomes in toxicity bioassays. In addition, we investigate the extent to which the available data can be used to predict the toxicity of untested NMPs based on the extracted parameters. Based on our results, we argue that focusing on a more diverse set of NMP properties combined with a more detailed characterization of the particles in future studies will help to fill current research gaps, improve predictive models and allow the identification of NMP properties linked to toxicity.

Nano- and microplastics: a comprehensive review on their exposure routes, translocation, and fate in humans.

Contamination of the environment with nano-and microplastic particles (NMPs) and its putative adverse effects on organisms, ecosystems, and human health is gaining increasing scientific and public attention. Various studies show that NMPs occur abundantly within the environment, leading to a high likelihood of human exposure to NMPs. Here, different exposure scenarios can occur. The most notable exposure routes of NMPs into the human body are via the airways and gastrointestinal tract (GIT) through inhalation or ingestion, but also via the skin due to the use of personal care products (PCPs) containing NMPs. Once NMPs have entered the human body, it is possible that they are translocated from the exposed organ to other body compartments. In our review article, we combine the current knowledge on the (1) exposure routes of NMPs to humans with the basic understanding of the potential (2) translocation mechanisms into human tissues and, consequently, their (3) fate within the human body. Regarding the (1) exposure routes, we reviewed the current knowledge on the occurrence of NMPs in food, beverages, personal care products and the air (focusing on indoors and workplaces) and found that the studies suggest an abundant presence of MPs within the exposure scenarios. The overall abundance of MPs in exposure matrices relevant to humans highlights the importance of understanding whether NMPs have the potential for tissue translocation. Therefore, we describe the current knowledge on the potential (2) translocation pathways of NMPs from the skin, GIT and respiratory systems to other body compartments. Here, particular attention was paid to how likely NMPs can translocate from the primary exposed organs to secondary organs due to naturally occurring defence mechanisms against tissue translocation. Based on the current understanding, we conclude that a dermal translocation of NMPs is rather unlikely. In contrast, small MPs and NPs can generally translocate from the GIT and respiratory system to other tissues. Thus, we reviewed the existing literature on the (3) fate of NMPs within the human body. Based on the current knowledge of the contamination of human exposure routes and the potential translocation mechanisms, we critically discuss the size of the detected particles reported in the fate studies. In some cases, the particles detected in human tissue samples exceed the size of a particle to overcome biological barriers allowing particle translocation into tissues. Therefore, we emphasize the importance of critically reading and discussing the presented results of NMP in human tissue samples.

Disentangling biological effects of primary nanoplastics from dispersion paints' additional compounds.

Microplastic particles (MP) and nanoplastic particles (NP) as persistent anthropogenic pollutants may impact environmental and human health. A relevant potential source of primary MP and NP is water-based dispersion paint which are commonly used in any household. Given the worldwide high application volume of dispersion paint and their diverse material composition MP and NP may enter the environment with unforeseeable consequences. In order to understand the relevance of these MP and NP from paint dispersion we investigated the components of two representative wall paints and analyzed their composition in detail. The different paint components were then investigated for their impact on the model organism Daphnia magna and on a murine cell line. Plastic NP, dissolved polymers, titanium dioxide NPs, and calcium carbonate MPs demonstrated adverse effects in both biological test systems, indicating detrimental consequences of several typical components of wall paints upon release into the environment. The outcome of this study may form the basis for the evaluation of impact on other organisms, environmental transport and impact, other related technical materials and for the development of strategies for the prevention of potential detrimental effects on organisms.

In-depth characterization revealed polymer type and chemical content specific effects of microplastic on Dreissena bugensis.

In aquatic ecosystems, filter feeders like mussels are particularly vulnerable to microplastics (MP). However, little is known about how the polymer type and the associated properties (like additives or remaining monomers) of MP impact organisms, as the predominant type of MP used for effect studies on the organismic level are micron grade polystyrene spheres, without considering their chemical composition. Therefore, we exposed the freshwater mussel Dreissena bugensis (D. bugensis) to in-depth characterized fragments in the same concentration and size range (20-120 µm): recycled polyethylene terephthalate from drinking bottles, polyamide, polystyrene, polylactic acid, and mussel shell fragments as natural particle control. Real-time valvometry, used to study behavioral responses via the movement of the mussels' valves, showed that mussels cannot distinguish between natural and MP particles, and therefore do not cease their filtration, as when exposed to dissolved pollutants. This unintentional ingestion led to polymer type-dependent adverse effects (activity of antioxidant enzymes and proteomic alterations), related to chemicals and residual monomers found in MP. Overall, recycled PET elicited the strongest negative effects, likely caused by anthranilamide, anthranilonitrile and butylated hydroxytoluene, contained in the fragments, which are toxic to aquatic organisms. As PET is among the most abundant MP in the environment, sublethal effects may gradually manifest at the population level, leading to irreversible ecosystem changes.

Taking advantage of transparency: A proof-of-principle for the analysis of the uptake of labeled microplastic particles by organisms of different functional feeding guilds using an adapted CUBIC protocol.

The analysis of the ingestion of microplastics (MP) by biota is frequently performed through invasive procedures such as chemical digestion protocols or by histological analysis of thin sections. Different, promising approaches for the observation of ingested MP particles pose so called tissue clearing methods. They are currently applied to organs, tissue samples, or whole organisms, rendering the sample transparent and enable to look inside an otherwise opaque environment. To date, there is a lack of methods to detect labeled MP inside an opaque organism's digestive tract without interfering with the sample's integrity. Therefore, our goal was to adapt the CUBIC tissue clearing protocol (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational Analysis) for aquatic and terrestrial organisms of various functional feeding groups for the analysis of the uptake of fluorescent labeled microplastic (MP) particles. We included the buff-tailed bumblebee Bombus terrestris, the compost worm Eisenia fetida, the woodlouse Porcellio scaber, the freshwater shrimp Gammarus roeselii, and the quagga mussel Dreissena bugensis in the analysis. The adapted CUBIC method has led to transparency in all normally opaque organisms. It further offers a simple way of locating fluorescent labeled MP inside the digestive system of the different organisms while leaving them intact.

Improving the proteome coverage of Daphnia magna - implications for future ecotoxicoproteomics studies.

Aquatic pollution is an increasing problem and requires extensive research efforts to understand associated consequences and to find suitable solutions. The crustacean Daphnia is a keystone species in lacustrine ecosystems by connecting primary producers with higher trophic levels. Therefore, Daphnia is perfectly suitable to investigate biological effects of freshwater pollution and is frequently used as an important model organism in ecotoxicology. The field of ecotoxicoproteomics has become increasingly prevalent, as proteins are important for an organism's physiology and respond rapidly to changing environmental conditions. However, one obstacle in proteome analysis of Daphnia is highly abundant proteins like vitellogenin, decreasing the analytical depth of proteome analysis. To improve proteome coverage in Daphnia, we established an easy-to-use procedure based on the LC-MS/MS of whole daphnids and the dissected Daphnia gut, which is the main tissue getting in contact with soluble and particulate pollutants, separately. Using a comprehensive spectral library, generated by gas-phase fractionation and a data-independent acquisition method, we identified 4621 and 5233 protein groups at high confidence (false discovery rate < 0.01) in Daphnia and Daphnia gut samples, respectively. By combining both datasets, a proteome coverage of 6027 proteins was achieved, demonstrating the effectiveness of our approach.

Shape, size, and polymer dependent effects of microplastics on Daphnia magna.

The effects of microplastic (MP) pollution on organisms are gaining increasing attention. To date, a variety of polymers of different shapes and sizes are used in ecotoxicology. Although polystyrene (PS) is the predominant polymer type used in effect studies, it is still unclear whether the observed effects derive from the polymer itself or from a certain particle shape and size. To elucidate whether the effects are polymer specific, we conducted a systematic study on Daphnia magna by comparing various PS-MPs to nonplastic control particles with similar properties. In chronic exposure experiments, we used PS beads (6 µm; 20 µm), fibers (Ø 3 µm, length: 75.5 µm), and fragments (5.7 µm; 17.7 µm) in two different size classes and two different concentrations (500 and 5000 particles ml-1) and in-house-produced control particles of comparable size, shape, concentration and, if possible, density. Although most PS properties did not elicit effects on the tested endpoints, we observed sublethal effects on D. magna life history and morphology for small PS beads and fragments. Interestingly, no adverse effects were detected for any of the control particles. Hence, the observed effects are polymer-specific, related to the size and shape of the polymer, and do not result from particle exposure per se.

Long-term exposure of Daphnia magna to polystyrene microplastic (PS-MP) leads to alterations of the proteome, morphology and life-history.

In the past years, the research focus on the effects of MP on aquatic organisms extended from marine systems towards freshwater systems. An important freshwater model organism in the MP field is the cladoceran Daphnia, which plays a central role in lacustrine ecosystems and has been established as a test organism in ecotoxicology. To investigate the effects of MP on Daphnia magna, we performed a chronic exposure experiment with polystyrene MP under strictly standardized conditions. Chronic exposure of D. magna to PS microparticles led to a significant reduction in body length and number of offspring. To shed light on underlying molecular mechanisms induced by microplastic ingestion in D. magna, we assessed the effects of PS-MP at the proteomic level, as proteins, e.g., enzymes, are especially relevant for an organism's physiology. Using a state-of-the-art mass spectrometry based approach, we were able to identify 28,696 different peptides, which could be assigned to 3784 different proteins. Using a customized bioinformatic workflow, we identified 41 proteins significantly altered in abundance (q-value <0.05) in the PS exposed D. magna. Among the proteins increased in the PS treated group were several sulfotransferases, involved in basic biochemical pathways, as well as GABA transaminase catalyzing the degradation of the neurotransmitter GABA. In the abundance decreased group, we found essential proteins such as the DNA-directed RNA polymerase subunit and other proteins connected to biotic and inorganic stress and reproduction. Strikingly, we further identified several digestive enzymes that are significantly downregulated in the PS treated animals, which could have interfered with the affected animal's nutrient supply. This may explain the altered morphological and life history traits of the PS exposed daphnids. Our results indicate that long-term exposure to PS microplastics, which are frequently detected in environmental samples, may affect the fitness of daphnids.